Initiation of Human DNA Replication in Vitro Using Nuclei from
Cells Arrested at an Initiation-competent State*
Received for publication, November 27, 1999, and in revised form, February 14, 2000
Torsten Krude‡
From the Wellcome/CRC Institute, University of Cambridge, Tennis Court Road, Cambridge CB2 1QR and the Department of Zoology, University of Cambridge, Cambridge,CB2 3EJ United Kingdom
Initiation of human DNA replication is investigated in vitro, using initiation-competent nuclei isolated from cells arrested in late G1phase by a 24-h treatment with
0.5 mM mimosine (Krude, T. (1999) Exp. Cell Res. 247, 148 –159). Nuclei isolated from mimosine-arrested HeLa cells initiate semiconservative DNA replication upon in-cubation in cytosolic extracts from proliferating human cells. Initiation occurs in the absence and presence of a nuclear membrane. The cyclin-dependent kinase (Cdk) inhibitors roscovitine and olomoucine inhibit initiation of DNA replication, indicating a dependence of initia-tion on Cdk activity. Cell fracinitia-tionainitia-tion shows that cyc-lins A, E, and Cdk2 are bound to nuclei from mimosine-arrested cells. Exogenously added human cyclin A䡠Cdk2 and cyclin E䡠Cdk2 complexes, but not cyclin B1/Cdk1 or cyclin D2/Cdk6, can overcome inhibition of initiation by roscovitine in vitro. Depleting Cdk2 from cytosolic ex-tract does not prevent initiation, demonstrating that cyclin䡠Cdk2 complexes are not required in the soluble extract, but are provided by the nuclei. Initiation de-pends further on an essential and soluble activity pres-ent in cytosolic extracts from proliferating cells, but not from mimosine-arrested cells, acting together with nu-clear cyclin/Cdk2 activity.
The initiation of DNA replication at the G1to S phase tran-sition is a key regulatory step of the cell division cycle in eukaryotic cells. Once DNA replication has initiated, control mechanisms ensure that the entire genomic DNA is replicated precisely once, and after completion, one replicated genome segregates to each of the two daughter cells during mitosis (for reviews, see Refs. 1– 6).
Cell fusion experiments in mammalian somatic cells estab-lished that G1, but not G2phase nuclei, initiate DNA replica-tion prematurely when exposed to an S phase cytosolic envi-ronment (7). Key regulators of initiation were identified in genetic and cytological experiments in vivo. Roles for cyclin-de-pendent protein kinases (Cdks)1 and their G
1 and S phase-specific regulatory subunits cyclin D, E, and A in inducing DNA replication have been documented (8 –16). The analysis of ini-tiation of DNA replication in vivo was recently complemented by a biochemical approach through the establishment of cell-free systems from human and mammalian cells (17–19). DNA replication is initiated in nuclei isolated from human G1, but not G2phase cells when incubated in S phase cytosolic extract
and S phase-specific nuclear factors. A nuclear extract could be substituted by purified recombinant cyclins A and E, com-plexed to their kinase partner Cdk2, to initiate DNA replica-tion, directly demonstrating functional roles for these nuclear cyclin䡠Cdk complexes (17). These cyclin/Cdks were essential, but not sufficient, as nuclei also required soluble factors present in a cytosolic extract from S phase cells to initiate replication (17).
For an assembly of replication-competent chromatin in eu-karyotic G1phase nuclei, an evolutionarily conserved series of molecular events is required involving the origin-recognition complex (ORC), Cdc6 protein, and the mini-chromosome main-tenance (MCM) proteins (reviewed in Refs. 2– 4, 20, and 21). In mammalian cell-free systems, competence to initiate DNA rep-lication in S phase cytosol is observed when template nuclei are prepared from late G1 phase cells after release from a block either in mitosis (17) or quiescence (18), followed by transit through early G1phase in vivo. Upon release from quiescence, competence of G1phase nuclei to initiate in vitro coincides with maximum expression of Cdc6 protein, and addition of recom-binant Cdc6 protein advances the onset of replication compe-tence in vitro (18). However, human cells undergoing mitotic proliferation contain Cdc6 protein at all stages of the cell cycle (22–24) and the state of DNA replication competence therefore depends on other factors as well.
Late G1phase nuclei from cells synchronized by release from either mitosis or quiescence are relatively undefined heteroge-neous and dynamic populations as a result of cells passing the state of competence at the time of preparation (17, 18). A significant step toward molecular and temporal dissection of the establishment of DNA replication forks in human somatic cells would therefore be the availability of defined populations of homogeneous template nuclei reversibly arrested in a state of replication initiation competence. A recently established cell-free system for the initiation of nuclear DNA replication in the yeast Saccharomyces cerevisiae made use of cells reversibly synchronized at defined points in the cell division cycle (25). Template nuclei from yeast mutants blocked at defined points in late G1phase initiated DNA replication at high percentages upon incubation in S phase nuclear extracts (25). Because such cell cycle mutants are currently not available as synchroniza-tion tools for human cells, I sought to achieve analogous arrest of human cells in late G1phase by chemical synchronization.
The plant amino acid mimosine is a versatile agent for block-ing DNA replication in proliferatblock-ing eukaryotic somatic cells but the literature on its mode of action is controversial. De-pending on cell type and concentration of mimosine, evidence for blocking both initiation and elongation steps of DNA repli-cation has been reported (26 –34). In the case of human cul-tured cells, however, this controversy has been clarified be-cause the different effects of mimosine depend on its concentration in the cell culture medium during treatment * This work was supported by the Royal Society and the Cancer
Research Campaign. The costs of publication of this article were de-frayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
‡ Royal Society University Research Fellow. Tel.: 44-1223-334109; Fax: 44-1223-334089; E-mail: [email protected].
1The abbreviation used is: Cdk, cyclin-dependent kinase.
© 2000 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.
This paper is available on line at http://www.jbc.org
13699
This is an Open Access article under the CC BY license.(34). At concentrations below 0.5 mM, mimosine interferes with elongation steps of DNA replication, and treatment results in populations of cells enriched in S phase after establishment of replication forks (34). In contrast, at concentrations of 0.5 mM and above, mimosine additionally prevents entry into S phase, resulting in a population of cells arrested in late G1 phase, before establishment of active DNA replication forks in vivo (34). Importantly, the late G1 phase arrest in vivo is fully reversible and cells enter S phase upon removal of mimosine (27, 34).
In this paper, I characterize the ability of nuclei from mi-mosine-arrested human cells to initiate DNA replication in human cell extracts. Nuclei prepared from these cells effi-ciently initiate semiconservative DNA replication upon incuba-tion in cytosolic extracts from proliferating cells, even in the absence of a nuclear membrane. Initiation depends on a cyto-solic extract from proliferating cells and, furthermore, on cyclin E/Cdk2 and/or cyclin A/Cdk2 activity which are present in the nuclei from mimosine-arrested cells. The data suggest that the competence of nuclei from mimosine-arrested cells to initiate replication is characterized by the presence of the cell cycle-regulated cyclin/Cdk2 proteins in cis and initiation is triggered by a different activity in trans.
MATERIALS AND METHODS
Cell Culture and Synchronization—HeLa-S3 cells were cultured as
monolayers and synchronized in G1, S, and G2phase exactly as
de-scribed (17). Synchronization in mitosis was performed as dede-scribed (35). Cells were arrested in late G1phase by adding 0.5 mMmimosine
(Sigma) from a 10 mMstock solution to proliferating cells for 24 h (34). Cell synchronization in interphase was determined by flow cytom-etry of isolated nuclei. One million nuclei were directly stained with propidium iodide (5g/ml in phosphate-buffered saline containing 0.4% Triton X-100) and analyzed by FACScan (Becton Dickinson) using the Lysis II-software. Data are presented as histograms showing relative DNA content (x axis) and cell number (y axis).
Preparation of Nuclei and Cell Extracts—Nuclei from
mimosine-arrested cells were prepared by hypotonic treatment, followed by Dounce homogenization and centrifugation exactly as described (34). Concentrations of nuclei were determined with a hemocytometer. Nu-clei were stored at ⫺80 °C for up to 3 months without loss of DNA replication competence. For permeabilization, nuclei were incubated in 0.1% Triton X-100 in SuNaSpBSA (250 mMsucrose, 75 mMNaCl, 0.5 mMspermine trihydrochloride, 0.15 mMspermidine tetrahydrochloride, 3% bovine serum albumin) at 4 °C in a rotator for 20 min and washed two times in SuNaSpBSA without Triton X-100.
Cytosolic and nuclear extracts from asynchronously proliferating and synchronized cells were prepared exactly as described (17, 35). Protein concentrations were determined with the Bio-Rad Protein assay using bovine serum albumin as standard. Cytosolic extracts were frozen in liquid nitrogen and stored up to 4 months at⫺80 °C without loss of replication initiation activity.
DNA Synthesis Reactions and Analysis of Reaction Products—DNA
replication initiation reactions (17) contained the following components: HeLa cell cytosolic extract (100g of protein, unless indicated other-wise); a buffered mixture of rNTPs and dNTPs including either biotin-16-dUTP (Roche Molecular Biochemicals) or [␣-32P]dATP as tracers
(17); and 2–5⫻ 105nuclei from mimosine-arrested HeLa cells (34). The
final reaction volume of 50l was adjusted with replication buffer (20 mMK-HEPES, pH 7.8, 100 mMpotassium acetate, 1 mMMgCl2, 0.1 mM
dithiothreitol). Where indicated, reactions were also supplemented with S phase nuclear extract (50g of protein). Incubation time was 3 h, unless indicated otherwise.
The Cdk-inhibitors roscovitine and olomoucine (both Calbiochem) were dissolved in dimethyl sulfoxide at 50 mM. When used, they were added to replication reactions at the final concentrations specified in the figure legends. Control reactions contained an equivalent volume of dimethyl sulfoxide.
Recombinant cyclin A/Cdk2 and cyclin E/Cdk2 were prepared from SF9 cells infected with recombinant baculovirus expression vectors (gifts of W. Krek, Friederich-Miescher-Institute, Basel). Purified hu-man cyclin B1/Cdc2 was a gift of M. Jackhu-man (Wellcome/CRC Institute, Cambridge) and cyclin D2/Cdk6 a gift of E. Laue and W. Zhang (De-partment of Biochemistry, University of Cambridge).
For analysis by confocal fluorescence microscopy, nuclei were fixed and spun onto coverslips. Total genomic and replicating DNA were visualized and analyzed exactly as described in Refs. 18 and 34. Anal-ysis of radioactively labeled replication products by acid precipitation and by density substitution were performed exactly as detailed before (Refs. 17, 18, and 34, and references therein).
Immunoblotting and Immunoprecipitation—Immunoblots of
cytoso-lic and nuclear extracts from HeLa cells were performed essentially as described (35). The following primary antibodies against human pro-teins were used: anti-Cdk1, anti-cyclin A, and anti-cyclin B1 (all gifts from J. Pines, Wellcome/CRC Institute, Cambridge); anti-cyclin D1 (ab171, AbCam); anti-Cdk2 (sc-163, Santa Cruz); anti-Cdk4 (sc-260, Santa Cruz); anti-cyclin E (HE12, a gift of Ed Harlow, Massachusetts General Hospital; sc-198, Santa Cruz).
Immunoprecipitation of cyclin E from human cell extracts was per-formed with polyclonal antibody sc-198 (Santa Cruz) from a total of 150 g of extract protein diluted in 1 ml of phosphate-buffered saline (36). The immunoprecipitate was washed in phosphate-buffered saline and subsequently analyzed by Western blotting using monoclonal antibody HE12.
Depletion of Cdk1 and 2 from Cytosolic Extract—Sepharose beads
coupled to human p9Cks1protein (37) (a gift of M. Jackman, Wellcome/
CRC Institute, Cambridge) were equilibrated and washed three times in replication buffer and concentrated by gravity sedimentation. Deple-tion of interphase cytosol was achieved by three successive rounds of (i) adding a fifth volume of the p9Cks1beads, (ii) incubating the slurry for
20 min at 4 °C in a rotator, and (iii) removal of the beads by pelleting at 13,000 rpm in an Eppendorf 5415C centrifuge for 5 min. Mock deple-tions were performed in parallel in the absence of p9Cks1-Sepharose.
RESULTS
Reversible G1Phase Arrest of Human Cells by Mimosine—
Asynchronously proliferating human cells arrest in late G1 phase when a 0.5 mM concentration of the plant amino acid mimosine is added to the culture medium for 24 h (34). Re-moval of mimosine from the culture medium in vivo results in a synchronous entry into S phase in about 50 –70% of the cells (27, 34). Our previous work has established that a proportion of late G1phase nuclei prepared from cells released either from mitosis (17) or quiescence (18) can serve as templates for ini-tiation of DNA replication upon incubation in human S phase extracts. Therefore, I investigated whether the mimosine-de-pendent reversible arrest point of human cells before entry into S phase could be exploited for a preparation of more homoge-neous populations of competent and defined template nuclei for efficient initiation of DNA replication in vitro.
Initiation of DNA Replication in S Phase Extracts—Nuclei
were isolated from mimosine-arrested human cells and used as templates for DNA replication reactions in vitro (Fig. 1). DNA synthesis was detected by incorporation of biotin-dUTP into genomic DNA and confocal fluorescence microscopy. Incubation of nuclei from mimosine-arrested cells in elongation buffer resulted in about 10% of the nuclei incorporating biotin-dUTP
in vitro (Fig. 1A). These nuclei represent a small proportion of
contaminating true S phase nuclei present in the preparation that continue semiconservative DNA replication at sites estab-lished prior to preparation in vivo (34). In contrast, addition of S phase cytosol to the reaction supported DNA synthesis in about 50 –55% of the nuclei (Fig. 1B). Because 10% of the nuclei had initiated DNA replication in vivo before preparation (cf. Fig. 1A and Ref. 34), this result demonstrates that S phase cytosol triggers initiation in about of 40 – 45% of the nuclei that had been arrested by mimosine before establishment of DNA replication forks in vivo. The percentage of nuclei synthesizing genomic DNA depended on the amount of S phase cytosolic protein added and reached saturation at 100g (Fig. 1C,
col-umns 2, 5, and 6). Addition of nuclear extract from S phase cells
also increased the percentage of nuclei synthesizing DNA (Fig. 1C, column 3). Addition of subsaturating amounts of both ex-tracts together increased the percentage of nuclei synthesizing DNA in vitro in an additive fashion (Fig. 1C, column 4). The
Initiation of Human DNA Replication in Vitro
maximum percentage of nuclei initiating DNA synthesis in
vitro was about 60 in most preparations, over and above the
contaminating proportion of S phase nuclei, corresponding to the percentage of cells entering S phase in vivo upon removal of mimosine.
The fluorescent signal of DNA synthesis in nuclei from mi-mosine-arrested cells was not homogeneous within the nuclei (Fig. 1B). Replicating nuclei were therefore analyzed by confo-cal microscopy at higher magnification (Fig. 1, D-F). Against the background staining of nuclear DNA (Fig. 1, D and F, red
signal), a clear pattern of many very small discrete
intranu-clear sites of DNA synthesis was detected (Fig. 1, E and F,
green signal). This pattern resembles the small replication foci
found in very early S phase, which are located in the euchro-matic regions of the nucleus (Fig. 1F; see Refs. 38 – 40, for reference). These data suggest that nuclei from mimosine-ar-rested cells initiate DNA synthesis in S phase extracts in vitro at sites used in early S phase in vivo.
In other eukaryotic DNA replication initiation systems, ac-cess of soluble initiation factors to the genomic DNA is regu-lated by the integrity of the nuclear membrane (1, 18). There-fore, I asked next whether removal of the nuclear membrane influences initiation of DNA synthesis in this system. Nuclei from mimosine-arrested HeLa cells were treated with 0.1% of the non-ionic detergent Triton X-100 to remove the nuclear membrane and used as templates for DNA replication in S phase cytosolic extracts (Fig. 2). Nuclear membrane permeabil-ity was measured by exclusion of fluorescent dextran. About 90% of the template nuclei were permeable, and remained permeable during the replication reaction in vitro (Fig. 2A). In comparison, without detergent treatment, typically 5–30% of the nuclei were permeable in a standard preparation by Dounce homogenization (data not shown). Importantly, about 40% of the permeable nuclei initiated DNA synthesis upon addition of S phase cytosol (Fig. 2B), which is the same
per-centage as compared with untreated nuclei (cf. Fig. 1C). There-fore, access of soluble initiation activity from S phase cytosolic extract to nuclei of mimosine-arrested cells is not influenced by the presence or absence of an intact nuclear membrane. These data establish that the terminal stages of initiation of human DNA synthesis in vitro as observed in nuclei from mimosine-arrested cells do not require the integrity of the nuclear mem-brane, consistent with observations made in extracts from
Xe-nopus eggs (41).
These data so far do not allow a distinction between initia-FIG. 2. Initiation of DNA replication in the absence of a nu-clear membrane. Nuclei from mimosine-arrested cells were perme-abilized with 0.1% Triton X-100. A, permeability of the nuclear mem-brane of treated nuclei before and after a 3-h replication reaction in
vitro. The permeability of the nuclear membrane was determined by
exclusion of fluorescent dextran using confocal microscopy. B, initiation of DNA synthesis in permeable nuclei from mimosine-arrested cells in S phase cytosolic extract. The percentages of nuclei replicating in the absence (white column) and the presence of S phase cytosol (black
column) were quantitated as described in the legend to Fig. 1.
FIG. 1. Initiation of DNA synthesis in nuclei from mimosine-arrested cells in S phase extracts. Nuclei were isolated from cells arrested in late G1phase by 0.5 mMmimosine (34) and incubated in elongation buffer (A) or S phase cytosol (B). Both incubations were in the presence of
nucleoside and deoxynucleoside triphosphates, including biotinylated dUTP, and were analyzed by confocal fluorescence microscopy. Nuclear DNA is visualized by propidium iodide (red signal) and DNA replication by fluorescein-conjugated streptavidin (green signal). Merged images are presented showing replicating nuclei in yellow-green and non-replicating nuclei in red. C, quantitation of the percentages of nuclei replicating in cytosolic and nuclear extracts from S phase cells. Replication reactions were performed in the presence of the indicated protein amounts of cytosolic (c) and nuclear (n) extract. The percentages of replicating nuclei were quantitated on microscopic fields containing 500 – 800 nuclei. D-F, high magnification analysis of a representative field of nuclei from mimosine-arrested cells incubated in S phase cytosol. Visualization of the propidium red channel (D, DNA), the fluorescein channel (E, replication), and the merged image of both channels (F) is presented. Scale bar, 10m.
tion of semiconservative DNA replication or DNA repair syn-thesis in vitro. Therefore, initiation of DNA synsyn-thesis was analyzed by density substitution (Fig. 3). Nuclei from mimo-sine-arrested cells synthesized only background amounts of DNA upon incubation in elongation buffer (34), migrating be-tween hemi- and unsubstituted densities (Fig. 3A). In contrast, incubation in S phase cytosol resulted in the synthesis of hemisubstituted DNA products (Fig. 3B). During this 2-h incu-bation in S phase cytosol, about 65 pmol of dNMP were incor-porated into 105replicating nuclei (data not shown), indicating that about 4 –5% of the genomic DNA was replicated. These data directly demonstrate that semiconservative DNA replica-tion is initiated in mimosine-arrested nuclei by a soluble activ-ity present in cytosolic S phase extract.
These data raise the possibility that this soluble initiation activity is inhibited by 0.5 mMmimosine in vivo, causing cell-cycle arrest before onset of S phase (34) and allowing efficient initiation of DNA replication in isolated nuclei upon incubation in S phase extracts. In the next experiments, I therefore char-acterized the competence of cytosolic extracts from mimosine-arrested cells to allow DNA replication in vitro.
Lack of DNA Replication Initiation Activity in Cytosolic Ex-tract from Mimosine-arrested Cells—First, S phase nuclei were
used as control templates for elongation of DNA replication in
vitro (Fig. 4A). As demonstrated before (34), S phase nuclei
elongate DNA replication at pre-existing replication forks in
either elongation buffer or in S phase cytosol (Fig. 4A, white and black columns). Cytosolic extract from mimosine-arrested cells allowed elongation in the same percentage of S phase nuclei (Fig. 4A, gray column), following the same incorporation kinetics as in S phase cytosol (data not shown). Density sub-stitution confirmed synthesis of hemisubstituted DNA prod-ucts in all of these three incubations (data not shown). These results demonstrate that cytosol from mimosine-arrested cells efficiently allows elongation of semiconservative DNA replica-tion at established active replicareplica-tion forks in vitro.
In contrast, cytosol from mimosine-arrested cells allowed initiation of DNA replication only in 15–20% of the nuclei from mimosine-arrested cells (Fig. 4B, gray column), over and above the background of S phase contaminants (Fig. 4B, white
col-umn). As S phase cytosol triggered initiation in about 50% of
these nuclei, the ability of cytosol from mimosine-arrested cells was significantly, but not entirely inhibited. This inhibition can either be explained by the presence of dominant inhibitors of initiation (but not of elongation), or the lack of soluble initiation activity in cytosol from mimosine-arrested cells. To discrimi-nate between these possibilities, I supplemented cytosolic ex-tract from mimosine-arrested cells with cytosol from G1and S phase cells (Fig. 4C). Addition of subsaturating amounts of either G1or S phase cytosol fully restored the initiation of DNA replication (Fig. 4C), demonstrating that cytosol from mimo-sine-arrested cells lacks soluble initiation activity.
The data of Fig. 4C suggest that cytosol from G1phase cells may also contain replication initiation activity. This would point toward a requirement for a different soluble initiation activity than the S phase-specific cyclin䡠Cdk complexes ob-served before, using nuclei from cells synchronized in late G1 phase by release from mitosis or quiescence (17, 18). I therefore analyzed the initiation activity of cytosolic extracts from cells arrested at all stages of the cell division cycle in relation to the presence of cyclin/Cdk proteins in these extracts.
Cell Cycle Specificity of Soluble DNA Replication Initiation Activity—HeLa cells were synchronized in early, mid, and late
G1phase, and in S, G2, and M phase (Fig. 5). The presence of cell cycle-regulated cyclins and their kinase partners in cyto-solic extracts from these cells was analyzed by Western blotting (Fig. 5A). Cyclin E protein was not detectable in early G1phase cytosol, but was present in maximum amounts in mid and late G1phase, and in lower amounts in S, G2, and M phase cytosol. Small amounts were also present in a salt extract from S phase nuclei (Fig. 5A, SN). Cyclin A protein was barely detectable in cytosol from G1phase cells, but was present in large amounts in S and G2phase cytosol. S phase nuclear extract contained
FIG. 3. Initiation of semiconservative DNA replication in
nu-clei from arrested cells in vitro. Nunu-clei from
mimosine-arrested HeLa cells were incubated in elongation buffer (A) and in S phase cytosol (B) in the presence of 5-bromodeoxy-UTP and [␣-32P]dATP for 2 h. Reaction products were analyzed by cesium
chlo-ride density equilibrium centrifugation as specified under “Materials and Methods.” The fraction numbers of the gradients and the positions of DNA not substituted (light-light, LL), hemisubstituted (heavy-light,
HL), and fully substituted with 5-bromodeoxyuridine (heavy-heavy, HH), as determined by refractive indices, are marked.
FIG. 4. Cytosol from mimosine-arrested cells lacks initiation activity. A, elongation of DNA replication by S phase nuclei in vitro. Template nuclei were prepared from S phase HeLa cells and incubated in the absence of cytosolic extracts (white column, labeled “buffer”) or in cytosolic extract (100g of protein) from mimosine-arrested cells (gray column, “mim”) and S phase cells (black column, “S”). The percentage of nuclei replicating was quantitated as specified in Fig. 1C. B, DNA replication in nuclei from mimosine-arrested cells in vitro. Nuclei from mimosine-arrested HeLa cells were incubated in vitro as specified in panel A. C, complementation of replication initiation activity in cytosol from mimosine-arrested cells by G1and S phase cytosolic extracts. Nuclei from mimosine-arrested HeLa cells were incubated in cytosolic extract from
mimosine-arrested cells, that was supplemented either with buffer only (white column, buffer), or with cytosolic extract (50g of protein) from mimosine-arrested (gray column, mim), S phase (black column, S), and G1phase cells (black column, G1).
Initiation of Human DNA Replication in Vitro
large amounts of cyclin A protein. Cyclin B1 protein peaked in G2 cytosol and was barely detectable in the other extracts. Cyclin D1 and the kinases Cdk1, Cdk2, and Cdk4 were clearly detectable in cytosolic extracts from all stages of the cell cycle. Additionally, Cdk2 was most abundant in S phase nuclear extract, similar to cyclin A (Fig. 5A, SN).
Next, the DNA replication initiation activity of these extracts was tested in vitro using nuclei from mimosine-arrested cells (Fig. 5B). Surprisingly, cytosolic extracts from all stages of G1 phase cells triggered initiation of DNA replication most effi-ciently in up to 60% of the nuclei. S and G2 phase cytosolic extracts were less active, triggering initiation in about 25– 40% of the nuclei and even cytosol of mitotic cells, arrested in metaphase by nocodazole, triggered initiation in about 25% of the nuclei. Initiation activity of S phase cytosol was stimulated by addition of S phase nuclear extract to the maximal efficiency observed in late G1phase cytosol alone (Fig. 5B).
These data clearly establish that the cytosolic DNA replica-tion initiareplica-tion activity for nuclei from mimosine-arrested cells is not strictly restricted to a particular phase of the cell cycle, however, it peaks in G1 phase and is partially inhibited in mitosis. Most importantly, replication initiation activity does
not simply correlate with the protein levels of the G1/S phase-specific cyclin E䡠Cdk2 and cyclin A䡠Cdk2 complexes present in these extracts.
These unexpected data raise two important questions, which I will address in the remaining experiments successively. (i) Does cytosol from untreated, asynchronously proliferating cells also trigger initiation in nuclei from mimosine-arrested cells? If so, this would provide an enormous simplification in the exper-imental protocol to study the initiation of human DNA replica-tion in vitro by dismissing the requirement to synchronize cells for preparation of initiating extracts. (ii) Is there a requirement for the G1/S phase-specific cyclin䡠Cdk complexes to initiate DNA replication in this system?
Interphase Cytosol Triggers Initiation of DNA Replication After a Short Lag Period—Nuclei from mimosine-arrested cells
were incubated in cytosolic extract from asynchronously prolif-erating cells and the time course of DNA replication was fol-lowed in vitro (Fig. 6). After an initial lag of 15 min, cytosol from interphase cells triggered efficient initiation of replication in about 30% of the nuclei during the following 45 min (Fig. 6A,
closed symbols). Then, the rate of initiation dropped to about
7% of nuclei initiating per hour for the remaining incubation. The amount of replicated DNA accumulated after an initial lag for at least 2 h (Fig. 6B, closed symbols). In control incubations in the absence of cytosolic extract, the background percentage of nuclei replicating did not change over the 3-h incubation period (Fig. 6A, open symbols) and only small amounts of DNA were synthesized (Fig. 6B, open symbols). These data demon-strate that interphase cytosol triggers initiation of DNA repli-cation in nuclei from mimosine-arrested cells efficiently after a short lag in the initial hour of the in vitro incubation. FIG. 5. Cytosol from all phases of the cell cycle allows
initia-tion of DNA replicainitia-tion in nuclei from mimosine-arrested cells. HeLa cells were synchronized in early G1(G1e, 4 h post-release from
nocodazole-arrest), mid G1(G1m, 6 h post-release), late G1(G1l, 8 h
post-release), S (2 h post-release from thymidine block) and G2phase (9
h post-release from thymidine block), and in mitotic metaphase (M, nocodazole-arrest) (17). Cytosolic extracts containing cytoplasmic and nucleosolic proteins were prepared from these cells. A high-salt extract from S phase nuclei (SN) was also prepared. A, Western blot analysis of
these extracts. For a detection of cyclin E protein, it was first immuno-precipitated from 150g of total extract protein and immunoprecipi-tates were analyzed by Western blotting. For the other proteins, iden-tical amounts of each extract (50g of protein) were directly analyzed by Western blotting using antibodies against the indicated human cyclin/Cdk proteins (see “Materials and Methods”). B, initiation of DNA synthesis in nuclei from mimosine-arrested cells in these extracts. Reactions contained identical amounts (100g) of the indicated cyto-solic extracts, and control reactions contained no cytosol (buffer, white
column) or both, S phase cytosol and 50g of protein of S phase nuclear extract (S⫹SN). The percentages of nuclei replicating were quantitated
as described in the legend to Fig. 1.
FIG. 6. Time course of initiation of DNA replication. Nuclei from mimosine-arrested cells were incubated either in the absence of cytosol (open symbols), or in cytosol from interphase cells (closed symbols) for the indicated times. A, quantitation of the percentages of nuclei repli-cating. The percentages of nuclei replicating was determined by confo-cal fluorescence microscopy as detailed in the legend to Fig. 1. B, quantitation of the amount of DNA synthesis. The amount of replicated DNA in these reactions was determined by [␣-32P]dATP incorporation
Initiation Depends on Cyclin/Cdk Activity—To address an
involvement of cyclin䡠Cdk complexes in the initiation of DNA replication in nuclei from mimosine-arrested cells, the influ-ence of the specific Cdk inhibitors roscovitine and olomoucine (42, 43) was first determined (Fig. 7). Concentrations above 0.5 and 2 mM roscovitine and olomoucine, respectively, inhibited replication to a background of about 10% of the nuclei (Fig. 7,
A and B). An inhibition to 50% of the maximal number of
replicating nuclei was observed at about 20Mroscovitine and 0.6 mMolomoucine (Fig. 7A), reproducing the 20-fold difference of the half-maximal inhibition by these two inhibitors of puri-fied Cdks 1, 2, and 5 but not of other kinases (42). In density substitution experiments, only small amounts of DNA synthe-sis products of intermediate densities between LL and HL were formed in the presence of 0.5 mM roscovitine (Fig. 7C). To-gether, these data strongly suggest that initiation of DNA replication in nuclei from mimosine-arrested cells is inhibited by roscovitine and olomoucine, and elongation of DNA replica-tion occurs only for relatively short distances in the 10% of contaminating S phase nuclei present in the preparation. The identity of the cyclin䡠Cdk complexes required for initiation was analyzed by adding recombinant human cyclin䡠Cdk complexes to reactions in the presence of roscovitine. Human cyclin A/Cdk2 and cyclin E/Cdk2, but not cyclin D2/Cdk6 or cyclin B1/Cdk1 could fully overcome the inhibition of initiation by roscovitine (Fig. 7D). These results strongly suggest an essen-tial and specific role for cyclin A/Cdk2 and/or cyclin E/Cdk2 activity in the initiation of DNA replication in this system. As the cytosolic initiation activity does not correlate with either cyclin A or E protein levels (Fig. 5), I therefore investigated the contribution of the kinase Cdk2 and the intracellular localiza-tion of the endogenous cyclin/Cdks to the initialocaliza-tion of DNA replication in this system.
Cyclin/Cdk2 Is Present in Cis and Is Not Required in Trans for Initiation—The contribution of the kinase Cdk2 from either
soluble extract in trans or nuclei in cis during the initiation reaction in vitro was first analyzed by depleting Cdks 1 and 2 from initiating interphase cytosol using Sepharose beads coated with protein p9Cks1(Fig. 8). Western blot analysis con-firmed depletion of Cdk1 and Cdk2 from the extract (Fig. 8A). As control, Cdk4 was not depleted (Fig. 8A). The depleted cytosol triggered initiation of DNA replication in nuclei from mimosine-arrested cells as efficiently as mock-depleted cytosol (Fig. 8B), indicating that Cdk2 (and Cdk1), and hence its ki-nase activity, is not supplied by the cytosol in trans in this system. Finally, the relative localization of these cyclin/Cdks in either cytosol or nuclei from mimosine-arrested cells was ana-lyzed by Western blotting (Fig. 8C). Cyclins A and E, and the kinase Cdk2 were clearly and predominantly present in salt extracts of nuclei from mimosine-arrested cells (Fig. 8C). Pro-tein levels of cyclin A and Cdk2 in cytosolic and nuclear ex-tracts from mimosine-arrested cells were similar to the protein levels found in cytosolic and nuclear extracts from S phase cells (data not shown). Taken together, these data demonstrate that cyclin A/Cdk2 and cyclin E/Cdk2 are provided by the nuclei in
cis. Therefore, initiation of DNA replication in nuclei from
mimosine-arrested cells requires nuclear cyclin/Cdk activity and an additional activity present in cytosolic extracts from untreated cells.
DISCUSSION
The experiments reported here demonstrated that 0.5 mM mimosine arrests human tissue culture cells in late G1phase at a state of competence to initiate DNA replication. Nuclei iso-lated from mimosine-arrested cells serve as efficient templates for initiation of DNA replication in soluble extracts from pro-liferating human cells. The state of competence to initiate DNA
replication correlates with a nuclear localization of cyclin A, E, and Cdk2 proteins. Initiation of DNA replication in vitro depends on nuclear cyclin/Cdk2 activity and an additional, essential and soluble initiation activity present in the cytosolic extracts.
FIG. 7. Initiation depends on cyclin/Cdk2 activity in vitro. A, inhibition of initiation in vitro by the Cdk inhibitors roscovitine and olomoucine. Nuclei from mimosine-arrested cells were incubated in interphase cytosol in the presence of the indicated concentrations of roscovitine (filled squares) and olomoucine (open squares). Percentages of nuclei replicating were determined by confocal fluorescence micros-copy as detailed in the legend to Fig. 1. B, representative field of replicating nuclei in the presence of 0.5 mMroscovitine (top panel, DNA; and bottom panel, replication). C, analysis of reaction products per-formed in the absence (open squares) and presence of 0.5 mMroscovitine (filled squares) by density substitution as specified in Fig. 3. D, rever-sion of the inhibition of initiation by recombinant human cyclin䡠Cdk complexes. Nuclei from mimosine-arrested cells were incubated in in-terphase cytosol in the presence of 0.5 mMroscovitine and 0.1– 0.2g of protein of the recombinant cyclin䡠Cdk complexes as indicated. Percent-ages of nuclei replicating were determined by confocal fluorescence microscopy as detailed in the legend to Fig. 1.
Initiation of Human DNA Replication in Vitro
Replication Initiation Competent Template Nuclei—Nuclei
from mammalian cells synchronized in the G1phase of the cell division cycle can initiate DNA replication upon incubation in human S phase extracts (17, 18). The competence of isolated
nuclei to initiate DNA replication in vitro arises in late G1 phase shortly before onset of S phase in vivo. Template nuclei were previously obtained from cells that are progressing through G1phase after a release from a block in either mitosis (17) or quiescence (18). As a result of the inherent degree of asynchrony of these dynamic cell populations, only limited and varying percentages of nuclei in a preparation initiate DNA replication in S phase extracts. However, using release from quiescence and addition of exogenous Cdc6 protein signifi-cantly increased the percentage of nuclei initiating in this approach (18), but the identification of endogenous markers for initiation competent nuclei is hampered by the relative asyn-chrony of the nuclear preparation.
Here, I report on the use of template nuclei prepared from human cells reversibly arrested in the cell cycle by 0.5 mM of the plant amino acid mimosine. These nuclei have a G1phase DNA content and do not contain active DNA replication forks capable of elongating DNA replication in elongation buffer in
vitro (Ref. 34 and this paper). As control, true S phase nuclei do
elongate DNA replication at existing forks under these condi-tions (Ref. 34 and this paper). Nuclei from mimosine-arrested cells initiate semiconservative DNA replication reproducibly with high efficiency upon incubation in cytosolic extracts from interphase cells (this paper).
Initiation competence of nuclei from mimosine-arrested cells correlates with high nuclear cyclin A/Cdk2 protein levels and initiation of DNA replication depends on nuclear, but not on cytosolic cyclin/Cdk2 activity (Fig. 8). In proliferating cells, cyclin A accumulates in the nucleus from S phase onwards until degradation in mitosis (44). Because of the nuclear local-ization of cyclin A, nuclei from mimosine-arrested cells could be considered S phase in character. However, active DNA replica-tion forks clearly have not been established in these nuclei (34), and by the stringent criterion of not synthesizing DNA, they have to be considered pre-S phase, or late G1phase in nature. It can therefore be concluded that mimosine blocks proliferat-ing cells in a state of initiation competence before the actual establishment of active DNA replication forks. It is also con-ceivable, that mimosine arrests human cells by preventing establishment of replication forks involving a late G1 phase checkpoint, but allowing continued cyclin A synthesis and nu-clear accumulation.
Nuclear membrane integrity is neither required for, nor in-hibits initiation of DNA replication in this system. This obser-vation is consistent with work on Xenopus egg extracts, where initiation of DNA replication can be observed in the absence of a nuclear membrane when chromatin is first incubated in cytosolic egg extract, followed by addition of a highly concen-trated nucleosolic extract (41). In this Xenopus system, repli-cation competent chromatin is assembled by the cytosolic egg extract and initiation is subsequently triggered by a high con-centration of soluble nuclear factors in the absence of an intact nuclear structure, mimicking a nuclear environment (41). In human cell extracts, it was demonstrated that an intact nu-clear membrane prevents exogenous Xenopus Cdc6 protein from binding to chromatin and establishing premature tion of DNA replication in mouse nuclei (18). However, initia-tion of DNA replicainitia-tion in the absence of exogenous Cdc6 protein was not increased or inhibited by permeabilizing the nuclear membrane at the beginning of the incubation in a subpopulation of template nuclei (18). The data reported here (Fig. 2) show that human nuclei from mimosine-arrested cells do not require, and are not inhibited by the nuclear membrane for initiation of DNA replication in human cell extracts. To-gether, these data suggest that G1phase events of establishing the competence of nuclei to initiate DNA replication may re-FIG. 8. Cis-requirement for cyclin A/Cdk2 and E/Cdk2 for the
initiation of DNA replication in nuclei from mimosine-arrested cells. A and B, cytosolic extract from interphase cells was depleted with p9Cks1
beads as specified under “Materials and Methods.” A, Western blot analysis of mock-treated (mock) and depleted cytosol (⌬). Identical protein amounts of each extract (50g) were separated on polyacryl-amide gels, blotted, and probed with antibodies against the indicated proteins as specified under “Materials and Methods.” B, DNA replica-tion in nuclei from mimosine-arrested cells upon incubareplica-tion in mock-treated and depleted cytosolic extracts. Nuclei from mimosine-arrested HeLa cells were incubated in vitro in the absence of cytosol (white
column, buffer) and in mock-treated and depleted cytosol (black col-umns, as indicated). The percentages of nuclei replicating were
deter-mined by confocal fluorescence microscopy as detailed in the legend to Fig. 1. C, localization of cyclins and Cdks in mimosine-arrested cells. Western blots of cytosolic and nuclear extracts (50g of protein each) from mimosine-arrested cells using antibodies against the indicated proteins as specified under “Materials and Methods.”
quire the assistance of selective transport across the nuclear membrane, however, initiation per se does not depend on it after competence for initiation is established.
The experimental approach of using template nuclei from mimosine-arrested cells provides an extensive and robust sim-plification in the experimental protocols to study human initi-ation of DNA repliciniti-ation in vitro. Prepariniti-ation of competent template nuclei involves a single synchronization step of pro-liferating human cells and trans-acting initiating extracts are obtained from unsynchronized proliferating human cells. This approach will allow future analysis of the molecular events during establishment of DNA replication forks in a variety of human cell types. Furthermore, it allows establishment of screening tests for novel inhibitors of the initiation of human DNA replication. This system, however, is limited in the anal-ysis of earlier G1 phase events during the establishment of competence to initiate DNA replication which lie before the arrest point of mimosine.
Involvement of Cyclin䡠Cdk Complexes in the Initiation of DNA Replication—Initiation of DNA replication in nuclei of
mimosine-arrested cells in vitro depends on the addition of cytosolic extract from interphase cells and requires cyclin A/Cdk2 or E/Cdk2 activity. The evidence for Cdk dependence stems from inhibition and rescue experiments using the spe-cific inhibitors roscovitine and olomoucine and is further sup-ported by correlating initiation activity with endogenous cyclin and Cdk proteins in the materials used.
Initiation of DNA replication in nuclei from mimosine-ar-rested cells was inhibited by roscovitine and olomoucine (Fig. 7). In human fibroblasts, both compounds arrest the cell cycle in G1 phase by inhibiting Cdk2, but not Cdk4 kinase (45). Specifically, both compounds inhibit purified Cdk1, 2, and 5 by competing with ATP binding at the active center of the kinases (42, 43, 46). The half-maximal inhibition (IC50) of purified protein kinase activity differs by a factor of about 20 between roscovitine and olomoucine (42). This relative difference in the IC50is also observed for the inhibition of DNA replication in nuclei from mimosine-arrested cells (Fig. 7), strongly arguing toward an essential requirement of cyclin䡠Cdk1/2 complexes in the initiation reaction. However, the absolute values of the IC50 differ between the two types of assay and can be explained by the presence of excess free ATP/Mg2⫹in the crude replication reactions. Lowering free ATP/Mg2⫹ in replication assays to levels used in the kinase assays with purified proteins (42, 43, 46) did not allow DNA replication to occur in vitro (data not shown). Therefore, the requirement for high ATP/Mg2⫹ pre-cluded determination of the IC50under ATP/Mg2⫹ concentra-tions of the kinase assays.
Specificity with respect to the kinase and its cyclin partner was analyzed by adding recombinant cyclin䡠Cdk complexes to
in vitro replication reactions in the presence of roscovitine (Fig.
7D). These experiments demonstrated that Cdk2 complexed to cyclin A and/or E could rescue the inhibition, supporting roles for one or both of these two kinases in initiating DNA replica-tion in human cell extracts (17).
However, the soluble initiation activity of cytosolic extracts from synchronized cells did not correlate with the endogenous protein levels of either cyclin A/Cdk2 or E/Cdk2 (Fig. 4). Cyto-solic cyclin A protein was present only in background amounts throughout G1phase and accumulated in S and G2phase (Fig. 4A, cf. Ref. 44). Cyclin E protein was absent in early G1but was induced maximally in mid/late G1phase and persisted at grad-ually decreasing concentrations through S and G2phase (Fig. 4A, cf. Refs. 13 and 47). Initiation activity was greatest in cytosolic extracts throughout G1 phase (Fig. 4B), indicating that protein levels of either or both cyclins in these extracts
cannot constitute the initiation activity of the cytosolic ex-tracts. The kinase Cdk2 was present in cytosolic extracts throughout the cell cycle and would therefore be available for association with the cyclin subunits to constitute protein ki-nase activity. However, a functional role for cytosolic cyclin A䡠Cdk2 and cyclin E䡠Cdk2 complexes in triggering initiation of DNA replication in nuclei from mimosine-arrested cells was directly excluded by depleting Cdk2 from interphase cytosol without loss of initiation activity (Fig. 8). Furthermore, addi-tion of purified recombinant human cyclin A/Cdk2 and cyclin E/Cdk2 to replication reactions in the absence of cytosolic ex-tract did not initiate DNA replication in nuclei from mimosine-arrested cells (data not shown). In any case, cyclin A/Cdk2, and to a lesser extent cyclin E, are provided in cis by the template nuclei from mimosine-arrested cells and could therefore consti-tute the roscovitine-sensitive initiation activity in vitro (Figs. 7 and 8). This nuclear localization of cyclins A and E, and Cdk2 in nuclei from mimosine-arrested cells may therefore also plain the lack of dependence on S phase-specific soluble ex-tracts to initiate DNA replication in this system.
In nuclei from human cells released from mitosis, cyclin A/Cdk2 and E/Cdk2 triggered initiation synergistically (17), whereas in nuclei from mimosine-arrested cells, both could overcome inhibition of initiation by roscovitine independently from each other (Fig. 7D). These data may indicate that a synergistic effect of both kinases is required for triggering initiation in nuclei from proliferating human synchronized at earlier stages of G1 phase before the mimosine-arrest point, and both kinases may act redundantly at later stages. How-ever, when nuclei from mouse cells released from quiescence were used as templates, only recombinant cyclin E/Cdk2, but not cyclin A/Cdk2 could overcome a block of initiation by olo-moucine (18). This suggests that the requirement for either cyclin䡠Cdk2 complex may actually vary with and depend on the synchronization procedures and sources of template nuclei used.
Mitotic cyclin B1 protein was enriched in G2 phase cytosol and was only present in background quantities in the other extracts (Fig. 4A), consistent with the intracellular localization of cyclin B1/Cdk1 in the human cell cycle (44, 48, 49). Cyclin B1/Cdk1 protein levels in the cytosolic extracts did not corre-late with the DNA replication initiation activity of these ex-tracts (Fig. 4) and cyclin B1/Cdk1 does not rescue inhibition of initiation of DNA replication by roscovitine. These data exclude a functional role for cyclin B1/Cdk1 in triggering initiation of DNA replication in human cells, consistent with previous data (17).
D type cyclins are expressed in response to mitogen stimu-lation (50) and, consequently, cyclin D1 and its kinase partner Cdk4 were found in all extracts of synchronized proliferating HeLa cells used here (Fig. 4A). This correlates with, and there-fore does not formally exclude an involvement of this soluble protein complex in triggering initiation of DNA replication. A direct functional role is, however, unlikely for the following reasons. Cyclin D/Cdk4 or 6 complexes are inhibited a 1000-fold less specifically by roscovitine or olomoucine than cyclin A or E䡠Cdk2 and cyclin B1䡠Cdk1 complexes (42, 43, 46). Further-more, recombinant human cyclin D2/Cdk6 did not rescue the inhibition of initiation of DNA replication by roscovitine (Fig. 7D). However, these data do not exclude the possibility that cyclin D complexes contribute indirectly to the initiation activ-ity present in interphase cytosol.
A Novel Soluble Initiation Activity—Taken together, the Cdk
dependence of initiation in nuclei from mimosine-arrested cells supports a model, where cyclin A/Cdk2 or E/Cdk2 are essential, but not sufficient for initiation of DNA replication. They are
Initiation of Human DNA Replication in Vitro
conferred by the template nuclei in cis. Initiation, furthermore, depends on an additional activity present in cytosolic extracts from untreated cells.
This model is supported by the observation that cytosol from mimosine-arrested cells lacks this soluble initiation activity (Fig. 5). However, this lack of initiation activity is not complete because initiation still occurs in about 10 –15% of the nuclei. This partial initiation deficiency is fully overridden by addition of small amounts of interphase cytosol from cells that are not arrested by mimosine, restoring full initiation activity (Fig. 5). This restoration of initiation activity can also explain the par-tial nature of initiation deficiency found in cytosol from mi-mosine-arrested cells by postulating the presence of residual, subsaturating initiation activity in the extract from mimosine-arrested cells. This residual activity can derive from the pro-portion of cells in the preparation, which are not at the arrest point, but in early/mid G1or S phase (34).
The results also suggest that the in vivo target of 0.5 mM mimosine preventing entry into S phase (34), could be identical to the soluble initiation activity found in the interphase cytosol from cells which are not treated with mimosine. Importantly, this initiation activity is not dramatically regulated throughout the cell cycle, however, it accumulates through G1phase and peaks before onset of S phase. The identity of this activity is currently unknown, but candidates may include one or more of activities like cyclin/Cdk activating factors, substrates for S phase-specific cyclin A/Cdk and E/Cdk kinases, or activities mediating the assembly of replication forks from DNA replica-tion proteins. We are currently purifying this soluble activity from cytosolic extract in order to identify factors that link cyclin/Cdk activity to the establishment of active DNA replica-tion forks in human cell nuclei.
Acknowledgments—I thank Ron Laskey, Jon Pines, Kai Stoeber,
Mark Jackman, David Szu¨ ts, and Heike Laman for discussions and critically reading this manuscript and Mark Jackman, Jon Pines, Ed Harlow, Ernest Laue, Wei Zhang, and Wilhelm Krek for antibodies and reagents.
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